Son is a recently identified nuclear speckle protein. Our long-term goal is to understand how nuclear speckles are assembled, how their dynamic structure is maintained, and how RNA processing factors are recruited from nuclear speckles to transcription sites where they function in pre-mRNA processing. Our hypothesis is that Son may play a novel role in maintaining nuclear speckle integrity and that Son may be essential for maintaining normal growth characteristics of cells. Based on Son's nuclear speckle localization, we also hypothesize that Son has a role in transcription and processing of pre-mRNAs. We base our hypotheses on our observations that 1) Son precisely colocalizes with pre-mRNA splicing factors in nuclear speckles;2) Son is a large protein with many novel repeat motifs that are not found in any other human protein;these sequences might support a novel role for Son as a scaffolding for RNA processing factors in nuclear speckles;3) Son depletion by siRNA treatment causes a disorganized speckle morphology and 4) Son depletion leads to a decrease in cell growth, cell viability and altered cell cycle progression. The experimental focus of this proposal is to use in vivo approaches and high-resolution microscopy techniques to study the functions of Son in the context of intact cells. Reported roles for Son in regulation of gene expression, repression of replication of Hepatitis B Virus, in the mitotic spindle apparatus and in anti-apoptotic response gives human disease relevance for studying Son functions. Specific aims of this project are to: 1. Establish a role for Son in maintaining nuclear speckle integrity. We will determine the regions of Son that dictate its nuclear speckle localization. We will examine changes in transcription and splicing in cells following Son depletion. We will use an inducible reporter gene locus to study dynamics of Son recruitment to a transcription site that will be used later on to map regions of Son required for the recruitment. We will also use siRNA rescue experiments to determine the regions of Son that are necessary to support nuclear speckle structure, normal cell growth characteristics, and pre-mRNA transcription and splicing. 2. Assess dynamics of Son and the effects of Son depletion on nuclear speckle dynamics during interphase and mitosis. We will examine the nuclear speckle dynamics of YFP-Son to determine if this putative structural maintenance protein shows less dynamic movements in interphase nuclei. We will also use live cell imaging techniques to determine the timing of Son recruitment to nuclear speckles in telophase nuclei. This timing will help us to decipher if Son nucleates speckle formation following mitosis. Public Health Relevance: The cellular location of Son in nuclear speckles makes it a likely candidate for gene regulation, which has been exemplified by the repressive activity of Son on the negative regulatory element (NRE) that lies upstream of the hepatitis B virus (HBV) core promoter leading to reduced replication and virus production (Sun et al., 2001). Son has also been described as an anti-apoptotic protein (Sun et al. 2001) and a mitotic spindle-associated protein (Nousiainen et al. 2006), and our own data indicates that Son depletion leads to defects in cell growth/survival and cell cycle progression as well as changes in nuclear speckle organization. Furthermore, the Son gene lies in head-to-head orientation with the glycinamide ribonucleotide formyltransferase (Gart) gene locus on chromosome 21, whose misregulation is suggested to be involved in leukemia in some Down's syndrome patients (Wynn et al., 2000). The implications for Son in regulating gene expression, in responding to cell death cues, and associating with mitotic spindle underscores the public health relevance for studying Son with regard to its functions in human diseases such as HBV viral infection and cancer.